A stem cell is an undifferentiated cell from which specialized cells are subsequently derived. Pluripotent stem cells can be differentiated into any of the three germ layers: endoderm, mesoderm, or ectoderm. Post fertilization, pluripotent stem cells form every cell type in the human body, including less plastic stem cell populations such as adult stem cells, fetal stem cells, and amniotic stem cells. Embryonic stem cells are a type of pluripotent stem cell, and possess extensive self-renewal capacity and pluripotency. More recently another type of pluripotent stem cell, induced pluripotent stem cells, were produced from mammalian terminally differentiated cells by a process termed somatic cell reprogramming. The process by which a stem cell changes into a more specialized cell is referred to as differentiation, for example the differentiation of endodermal progenitor cells to hepatocytes.
Stem cell derived somatic cells represent a large source of cells for basic and translational science. While promising, there are many hurdles that need to be overcome to make this a reality. In particular, current protocols produce hepatocytes of variable function and longevity which is the product of using undefined components in the differentiation process. The use of Matrigel® and serum are prime examples, and remains a source of batch to batch variability in hepatocyte differentiation procedures. Matrigel® is a complex tumor and BM-like extract obtained from murine Engelbreth-Holm-Swarm (EHS) sarcoma tumor tissues. Matrigel® mainly contains murine LN-111, type IV collagen, perlecan and nidogen but also varying amounts of other materials, including growth factors and cellular proteins and, therefore, its composition is undefined and varies from batch-to-batch. More recent studies have used small molecules to replace growth factors, but while this dramatically drives down process costs, small molecules can have some off-target effects. Moreover those studies used Matrigel® and serum to drive the differentiation process, and by nature will therefore be variable. As a result, generating reliable, reproducible cultures of hepatocytes is difficult. Moreover, if these cells are to be used clinically, manufacturing processes must meet GMP guidelines. To comply with these guidelines, products containing animal derivatives are strictly controlled.
It would be desirable to develop methods that allow for differentiation of stem cells under chemically defined, xeno-free, pathogen-free, and stable batch-to-batch conditions into differentiated cells, particularly hepatocytes. Desirably, such methods should provide large quantities of such differentiated cells from GMP grade human embryonic stem cells (hESCs).